This suggested alternative sites of reactivity with AA. Carbon-4 may be the preferred site for AA-pigment interactions, but it was not the only location. With Carbon-4 blocked, 5-Carboxypyranocyanidin-3-galactoside reacted with AA to form new pigments and reduce bleaching.Juices colored by anthocyanins experience color loss related to fortification with ascorbic acid (AA), thought to be the result of condensation at Carbon-4 of anthocyanins. To further understand this mechanism, pyranoanthocyanins, having a fourth-ring covalently occupying Carbon-4, were synthesized to compare its reactivity with AA against that of anthocyanins. Pyranoanthocyanins were synthesized by combining chokeberry anthocyanins with pyruvic acid. AA (250–1000 mg/L) was added to either chokeberry extract, cyanidin-3-galactoside, or 5-Carboxypyranocyanidin-3-galactoside. Samples were stored in the dark for 5 days at 25 °C and spectra (380–700 nm), color (CIE-L*c*h*), and composition changes (HPLC-MS/MS) were monitored. Extensive bleaching occurred for cyanidin-3-galactoside and chokeberry colored solutions, with a decrease in half-lives from 22.8 to 0.3 days for Cyanidin-3-galactoside when 1000 mg/L AA was added. 5-Carboxypyranocyanidin-3-galactoside solution better maintained color with limited loss in absorbance, due to the formation of colored degradation products (λ Consumers commonly use color to make assessments on acceptance and liking, implied flavor, safety, and overall quality of food products . SynthetAnthocyanins (ACN) are a class of water-soluble polyphenols found in many fruits and vegetables. Their color properties are greatly influenced by the substitution patterns on the aglycone structure as well as pH environment . Warm huIt has long been known that the presence of AA in anthocyanin-colored solutions can accelerate degradation and loss of color . BisulfiPrevious work has found that anthocyanins with both 3- and 5-substitions increase pigment stability against ascorbic acid compared to just 3-substitution, likely a result of further restricting access to C4 in between. Viguera and Bridle reported that Malvidin-3,5-diglucoside experienced slower color loss as compared to Malvidin-3-glucoside. The same authors reported that direct substitution of the C4 with phenyl and methyl groups enhanced their stability against ascorbic acid color loss versus typical –H substitution . CopigmePyranoanthocyanins  are formThe objective of this study was to compare the reactivity of anthocyanins and pyranoanthocyanins upon the addition of AA. If anthocyanin bleaching with AA occurs only at site C4, pyranoanthocyanins, with C4 unavailable, would not undergo bleaching. Furthermore, no changes would occur on the PACN pigments as a results of AA addition. Our hypothesis was that C4 is not the only site of reactivity. Comparison of the reactivity of these pigments in response to ascorbic acid will aid in further understanding the deleterious interaction of this micronutrient and pigment and could allow for the better selection of anthocyanin sources in applications with ascorbic acid.p-values of less than 0.01. As AA levels increased, the loss in absorbance for each pigment over time also increased, revealing a dose-dependent effect of AA. For the 500 mg/L AA treatments over a 5-day period, 5-Carboxypyranocyanidin-3-galactoside saw a reduction of 38% reduction in maximum absorbance, chokeberry a 79% reduction, and Cyandin-3-galactoside an 88% reduction.Anthocyanins degraded quickly in the presence of ascorbic acid, as seen in vis-max were also observed over the 5-day period. For the 500 mg/L AA level, the following hypsochromic changes in λvis-max occurred: chokeberry, 512 to 511 nm; cyanidin-3-galactoside, 511 to 509 nm, 5-Carboxypyranocyanidin-3-galactoside, 491 to 484 nm. These shifts in λvis-max are reflected in vis-max observed over the 5 days of the AA treatment were less than 5 nm, regardless of the levels of AA, and were more likely attributed to pigment degradation. The PACN experienced hypsochromic shifts as large as 10 nm with less loss in maximum absorbance, with shifts becoming more pronounced as AA levels increased.Changes in λvis-max over time. Bleaching has been previously reported as a first-order reaction, typical of ACN degradation, and was modeled as such in determining the reaction rate and half-life [L*) as well as the decrease in chroma (c*) for all pigments. The reduction in maximum absorbance closely followed first-order kinetics for all treatments with an R2 higher than 0.94 for control treatments and 0.96 for samples with added ascorbic acid. The kinetics results for each pigment and AA level can be found in Degradation kinetics were evaluated in terms of change in maximum absorbance of the solutions at the original λalf-life ,25. The This order of stability was also exhibited across all AA levels. The enhanced stability and extension of half-life for 5-Carboxypyranocyanidin-3-galactoside was more evident upon the addition of ascorbic acid. Pyranoanthocyanins exhibited a half-life 8–13x higher than Cyanidin-3-galactoside in the presence of AA. Kinetics data supports the hypothesis that C4 is likely the primary or preferred site for anthocyanin-ascorbic acid interaction. It also reveals that the pyranoanthocyanin was still susceptible to ascorbic acid through alternative mechanisms or sites due to the half-life lowering in response to AA compared to its control.2 values for these pigments at varying AA levels are the following: chokeberry extract, 0.99; Cyanidin-3-galactoside, 0.96; and 5-carboxypyranocyanidin-3-galactoside, 0.98. Linearity is lost to some degree for Cyanidin-3-galactoside with 1000 mg/L AA addition. The slope could be effectively regarded as how responsive (or deleterious) the change in pigment solution maximum absorbance is upon AA addition, with a higher slope indicating greater susceptibility to AA. The slope of Cyanidin-3-galactoside was 3.1× that of chokeberry extract and, in comparison to 5-carboxypyranocyanidin-3-galactoside, cyanidin-3-galactoside was 8.2× and chokeberry extract 2.7× more suspectible.The relationship between ascorbic acid level and the reaction rates was additionally assessed to observe how each of these pigments responds to ascorbic acid addition. A linear relationship was found and can be seen in L*) in L* increased from 77.2 to 96.4 (∆19.2) for Cyanidin-3-galactoside; chokeberry, 74.4 to 89.4 (∆15.0); and 5-Carboxypyranocyanidin-3-galactoside, 81.7 to 86.6 (∆4.9). The presence of AA resulted in higher lightness over time, and this was dose-dependent. Pyranoanthocyanins showed the least change in L* in reponse to AA, and Cyanidin-3-galactoside the greatest. An increase in L* represents a lighter color expression and was most evident for chokeberry and cyanidin-3-galactoside.Rapid color loss and extensive bleaching of pigments can be seen with CIE lightness , a more orange-red hue than Cyanidin-3-galactoside (16.6°), with a more red color  were calculated as the color change from day 0 to 5 for each respective treatment, and are presented in vis-max of 322 nm was likely chlorogenic acid, as it is reported to be present in the berry [To determine the relationship between spectral and color changes with changes in pigment composition, HPLC analysis was utilized. The initial (day 0) HPLC profile for chokeberry extract revealed the following anthocyanin profile seen in he berry . After 1vis-max: A, 487 nm; B, 486 nm; and C, 477 nm. The formation of these new compounds aligned with both colorimetric data (increase in hue angle) and the spectral shift (hypsochromic response) that was observed with the solutions in response to AA. The formation of the new peaks is likely how the pyranoanthocyanin solution better maintained original color expression even with the rapid loss of the parent compound. The formation of three new chromophores between the PACN-AA interaction could be the result of several different phenomena, and additional experiments were performed to include MS/MS data of the new structures.For the pyranoanthocyanin, the isolated structure accounted for 94% of the AUC from the maxplot. By day 1, this peak was reduced 93% and 99% by day 5; however, unlike the anthocyanins, where the pigments degraded into colorless forms, the PACN-AA interaction resulted in the development of new peaks in the visible range, labeled A, B, and C in For the three peaks produced after AA addition, it is possible that these are the result of interaction with PACN at alternative sites (not C2 or C4). With the addition of a fourth ring, ascorbic acid could have reacted with the D-ring substitution (carboxylic acid group) and produced colored byproducts. It has previously been reported that acetyl pyranoanthocyanins experience both a hyperchromic and hypsochromic shift in response to up to 200 ppm sulfites, and it was proposed this was the result of sulfite covalent linkage at the acetyl group in the D-ring, enhancing the molar absorptivity .m/z of 535. This was +18 mass units (m.u.) compared to 5-carboxypyranocyanidin-3-galactoside. A followup product ion scan revealed a daughter ion of 373 m/z, a transition of −162 m.u. from the parent ion. This is a commonly reported transition for glycosylated molecules and matches the loss of galactose from the structure [m/z of 535, direct condensation of ascorbic or dehydroascorbic was ruled out. Ascorbic acid degradation byproducts were also considered. Ascorbic acid has previously been reported as being catalyzed by trace levels of metal (1 μM) to form hydrogen peroxide [m/z being +16 mass units. A different mechanism probably occurs, possibly related to the condensation of the pyranoanthocynin with other AA degradation products, and perhaps even the rearomatization of the molecule.The MS/MS data generated provided valuable insight to the structures of the newly formed compounds. Peak A was the only structure that was not present in trace amounts in the control treatment. A positive ion scan revealed a parent tructure . This superoxide ,29,30. Aperoxide  reportedm/z of 519, and the respective product ion scan revealed a daughter ion at 357 mass units. Peak C had a parent m/z of 503 and the product ion scan revealed a daughter with 341 mass units The shifts for all structures was −162 m.u. from parent to daughter ion, which was again attributed to the loss of galactose. It is important to point out that the m/z of peak C was lower than that of our starting pyranoanthocyanin , with a loss of 14 units. Elucidation of these chemical structures will require NMR work, currently underway.Peak B, under positive ionization, had a parent m/z, respectively) did not appear in the chromatogram. This suggests that the newly formed peaks are not the result of the direct condensation of ascorbic and dehydroascorbic acids with the pyranoanthocyanin.Of the three new compounds, the structural modification induced by ascorbic acid addtion was isolated to the aglycone. Interestingly, single ion monitoring for possible direct condensation products of ascorbic or dehydroascorbic acid with 5-carboxypyranocyanidin-3-galactoside  was purchased from Sigma Aldrich. HPLC grade acetonitrile and water were obtained from Fisher Scientific  and HPLC grade formic acid from Sigma Aldrich.Powdered chokeberry fruit was provided by Artemis Inc. . Lab grade pyruvic acid used for the synthesis of pyranoanthocyanins was purchased from Sigma Aldrich . Analytical grade ascorbic acid (99% Chokeberry powder was mixed with water acidified with 0.01% HCl prior to purification. The solution was loaded onto a Waters Sep-pak C18 cartridge for solid phase extraction (SPE). The column was then washed with acidified water (0.01% HCl) to remove sugars and acids, followed by washing with ethyl acetate for the removal of the more non-polar phenolics. Pigments were recovered from the cartridge with methanol acidified with 0.01% HCl, and the solvent was removed by rotary evaporation . Pigments were then solubilized and stored in acidified water for future use. This was the only preparatory step for chokeberry treatments.Pyrananthocyanins were synthesized from the semi-purified chokeberry by the addition of pyruvic acid. The extract  was added to a pH 2.6 citrate buffer that had 0.1% potassium sorbate and 0.1% sodium benzoate to prevent molding. A molar ratio of 1:50 (ACN: pyruvic acid) was followed as previously described . The prev/v) with Solvent A being 4.5% formic acid in HPLC grade water and Solvent B was HPLC grade acetonitrile. Elution of peaks was monitored at 500 nm. Peaks were manually collected. The two collected peaks were diluted with distilled water and again subjected to SPE semi-purification to remove formic acid and acetonitrile. Rotary evaporation was used to remove methanol , and the pigments were stored in 0.01% HCl in acidified water.A reverse phase HPLC system composed of the following modules was used: LC-6AD pumps, CBM-20A communication module, SIL-20A HT autosampler, CTO-20A column oven, and SPD-M20A Photodiode Array detector . The reverse-phase column selected was a 250 × 21.2 mm Luna pentafluorophenyl column with 5 µm particle size and 100 Å pore size . Samples were filtered prior to injection with a Phenex RC 0.45 µm, 15 mm membrane syringe filter . With a flow rate of 10 mL/min and a run time of 30 min, peaks were separated and collected. An isocratic system with the following solvents were used: 11:89 . Purified pigments were filtered using Phenex RC 0.45-µm membranes. A binary system with 1 mL/min flow rate was used: solvent consisted of 4.5% formic acid in HPLC grade water and solvent B consisted of HPLC grade acetonitrile. The gradient began with an isocratic flow of 6% solvent B for 17 min (elution of primary anthocyanins), increasing to 15% solvent B by 45 min (elution of primary pyranoanthocyanin), and to 40% solvent B by 50 min (wash). A 10-µL injection volume was loaded onto a Phenomenex Kinetix 5-µm EVO C18 100 A. 150 × 4.6 mm column with a Phenomenex Ultra UHPLC EVO C18 guard cartridge attached. Purity was expressed in terms of % peak area of targeted pigment as compared to the total area of all peaks present in the max plot (260–700 nm). The isolate of 5-Carboxypyranocyanidin-3-galactoside accounted for 94% of the overall area under the curve (AUC), cyanidin-3-galactoside isolate was 92% AUC, while chokeberry ACN purity was 35% AUC. Chokeberry ACN likely contained other phenols present in the source material.vis-max was reached. Levels of AA of 250, 500, and 1000 mg/L were added using a concentrated ascorbic acid stock solution, and a control consisting of each pigment with the absence of AA was maintained. All samples were brought to the same final volume with additional citrate buffer. The pH of all samples were evaluted using a S220 SevenCompact pH meter  and were found to have a pH of 3.0 ± 0.05. Samples were stored in the dark at 25 °C in an incubator  until an absorbance of 1.0 at their respective λd-lysine coated polystyrene) were used for the evaluation of absorbance from 380–700 nm, at 1-nm intervals. Aliquots (200 µL) of samples were loaded into individual wells, and a blank consisted of the same citrate buffer used.A SpectraMax 190 Microplate Reader  with a 96-well plate (poly-ColorBySpectra) written for color conversion, absorbance data was translated to the CIE  L*c*h color space [Using UV-Vis spectral data  in combination with software , and 520 nm . A max plot of 470–520 nm was later added to standardize changes in the AUC for all three pigments and their degradation products.Prepared solutions were monitored to determine the formation of potential degradation products or profile changes. Using the analytical HPLC system and conditions previously described , chromatm/z; product ion scan, 519 m/z; product ion scan, 503 m/z; and precursor ion scan, 355 m/z. These secondary collision events all had an event time of 0.1 s and a collision energy of −35.0 eV. To also consider the possibility of condensation of the pyranoanthocyanin and ascorbic acid, the following single ion monitoring was also included: 675 m/z for 5-carboxypyranocyanidin-3-galactoside (517) + ascorbic acid (176) − H2O (18) and 673 m/z for dehydroscorbic acid (174).With the development of newly formed peaks for the pyranoanthocyanin, tandem mass spectrometry (MS/MS) was performed to obtain additional structural information on the novel structures. The same HPLC conditions and column mentioned in Data was organized for means and standard deviations using Microsoft Excel . One-way ANOVA was performed for each treatment at all time points to determine if a significant change in CIE-L*c*h and maximum absorbance occurred. One-way ANOVA (two-tailed) was also performed for each pigment  at each time point to determine if and when which CIE-L*c*h and maximum absorbance became significantly different from the control. Software used for ANOVA tests was SPSS .Cyanidin-3-galactoside degraded rapidly in the presence of ascorbic acid, followed by chokeberry extract. Other phenols in chokeberry extract likely played a protective role against AA-mediated pigment bleaching. The 5-Carboxypyranocyanidin-3-galactoside colored solution exhibited the smallest change in color (ΔE) and limited bleaching in response to ascorbic acid . The interaction between PACN-AA resulted in the formation of three new chromophores, as revealed by HPLC. NMR work is underway to determine the site of reaction for PACN-AA as well as the ACN-AA reactivity. The fact that PACN, with the C4 position blocked, still exhibited limited bleaching further supports the hypothesis that C4 plays an important, but not singular, role in the AA-mediated bleaching of anthocyanins. The pyranoanthocyanin better maintained absorbance and color expression in the presence of AA, not as a result of 5-Carboxypyranocyandin-3-galactoside survival, but due to the formation of colored byproducts suspected at alternative sites.


O-rutinoside, and luteolin-7-O-glucoside. Minor quantities of cynarin, luteolin, apigenin-7-O-glucoside, apigenin-7-O-rutinoside, and naringenin-7-O-glucoside were also found. The antioxidant activity of the obtained extracts determined by ABTS , DPPH , and FRAP (Ferric Ion Reducing Antioxidant Power) was highly correlated with the total concentration of phenolic compounds. Chlorogenic acid, luteolin-7-O-glucoside, and luteolin-7-O-rutinoside, the most abundant compounds in 60% methanol extracts, are the components most responsible for the antioxidant activity of the artichoke waste extracts. The extract with the best antioxidant capacity was selected to assay its antioxidant potential on a model intestinal barrier. This action of the hydroxycinnamic acids on intestinal cells (Caco-2) was confirmed. In summary, artichoke waste may be considered a very interesting ingredient for food functionalization and for therapeutic purposes. Artichoke waste represents a huge amount of discarded material. This study presents the by-products  of the “Blanca de Tudela” artichoke variety as a potential source of phenolic compounds with promising antioxidant properties. Artichoke residues were subjected to different extraction processes, and the antioxidant capacity and phenolic composition of the extracts were analyzed by spectrophotometric methods and high performance liquid chromatography (HPLC) analyses, respectively. The most abundant polyphenols in artichoke waste were chlorogenic acid, luteolin-7- The agri-food industry generates millions of tons of waste each year, which leads to serious environmental problems and a great loss of very valuable biocompounds . VegetabCynara scolymus L., family Asteraceae) is a herbaceous perennial plant which originated in the Mediterranean area, but which is now widely grown all over the world. France, Spain, and Italy comprise 80% of the total worldwide production and the wide consumption of artichoke makes it an important produce for these Mediterranean countries. In artichokes, the edible parts of the plant are large immature inflorescences, named capitula or heads. During the industrial processing of artichoke, about 60–85% of harvested vegetal is discarded  used is based on the method outlined by Re et al. . The calThe DPPH assay  used is based on the method outlined by Brand-Williams et al. . For theThe antioxidant capacity of extracts was also determined by the FRAP assay proposed by Benzie and Strain . Known cFinally, the total phenolic content (TPC) was analyzed using the Folin–Ciocalteu method outlined by Singleton et al. . Briefly2 at 37 ºC. Cells (passages 20–50) were grown in Dulbecco’s Modified Eagles medium (DMEM)  supplemented with 20% fetal bovine serum, 1% non-essential amino acids, 1% penicillin (1000 U/mL), 1% streptomycin (1000 μg/mL), and 1% amphotericin (250 μg/mL). Cell medium was replaced every three days and cells were passaged once a week with 0.25% trypsin-1 mM EDTA. Cell confluence (80%)—differentiated cells—was confirmed by microscopic observance. The human colorectal adenocarcinoma Caco-2 cell line (TC7 clone) was kindly provided by Dr. Edith Brot-Laroche . Cells were maintained in a humidified atmosphere of 5% CO50 was defined as the concentration of extract that reduced cell viability to 50%.Cells were seeded on 96-wells plates at a density of 4000 cells per well and they were incubated under standard culture conditions until differentiation (7 days). Cell confluence (80%) was confirmed by microscopic observance. For treatment, lyophilized fractions 1 and 2 from extracts obtained with methanol:water 60% (v/v) and ultrasounds were weighed and dissolved in cell culture medium  to the required concentrations: 1000, 500, 250, 125, and 62.5 μg/mL. These concentrations were chosen in relation to previous studies in our laboratory . Cells w2O2 (dissolved on PBS: Phosphate Buffer Solution) was added to each well except negative control cells, which were incubated with cell culture medium. After 20 minutes incubation with H2O2 protected from light, the fluorescence intensity was measured using a FLUOstar Omega Microplate Reader . Excitation and emission wavelength settings were 485 and 535 nm, respectively. The intensity of fluorescence is considered as a reflection of the total intracellular ROS levels.Cells were seeded on 96-well plates at a density of 4000 cells per well and were incubated under standard culture conditions until differentiation (7 days). For treatment, extracts were dissolved on cell culture medium  to the required concentrations: 1000, 500, and 250 μg/mL. Cells were incubated with the extracts for 1 or 2 h. Then, cells were incubated for 20 min with 20 mM 2′,7’-dichlorofluorescein diacetate (DCFH-DA) at 37 °C protected from light, according to the assay previously described by Sánchez-de-Diego et al. . For hydData are presented as the mean ± SE. Data were subjected to a two-tailed t-test and differences were considered significant at p ≤ 0.05. Statistical analysis was carried out using IBM SPSS software v 23 . 2+, but the oxidant conditions of the medium induce the formation of a complex of chlorogenic acid/Fe3+ gray-blue, responsible for the artichoke browning [browning . Cynarinbrowning ,32. O-rutinoside and luteolin-7-O-glucoside, while very small amounts of the rest of the flavonoids were found  was not detected in the extracts obtained using 60% methanol as the extraction solvent, although when water was used in the extraction, apigenin-7-O-glucoside was the flavonoid that was not detected in the extracts. It is clear that the solvent type significantly affected phenolic compounds recovery. Differences in the extraction efficiency are mainly due to the higher range of polarity of 60% methanol as a solvent compared with 100% water, but other factors, such as weakening of the solute–matrix interactions and swelling of the plant material, could also be involved [O-glucoside provide beneficial effects for health due to their anti-cancerous and antioxidant properties [O-glucoside [The most abundant flavonoids in the extracts obtained from artichoke waste using 60% methanol were luteolin-7-re found . These rcuronide ,21,33. Ninvolved . Narirutoperties . The thelucoside ,37. Hydrlucoside , but it lucoside ,38 and hThe ultrasound application only increased the content of phenolic compounds when 60% methanol was used as the solvent. In contrast, Punzi et al.  found thAs far as artichoke phenolics are concerned, much of the research has focused on the antioxidant activity of artichoke leaf extracts, but in this research work, it has been shown that artichoke processing waste may constitute an alternative to the commercial leaf extracts currently available for liver care and cholesterol metabolism.O-glucoside, and luteolin-7-O-rutinoside, the most abundant compounds in 60% methanol extracts, are the components most responsible for the antioxidant activity of the artichoke waste extracts. Additionally, Fritsche et al. [The antioxidant capacity of the different extracts obtained from artichoke waste was evaluated by the ABTS, DPPH, and FRAP methods. It is important to properly assay the antioxidant capacity of food-derived extracts because it is due to a combination of the activities of several antioxidant compounds. e et al.  found the et al. ,32,45,46e et al. . Conseque et al. . p < 0.05) correlation with the content of luteolin-7-O-rutinoside (Pearson R of 0.962), the most abundant flavonoid in our artichoke waste extracts. The antioxidant capacity of the total extract was much greater than the sum of the antioxidant capacities of fractions 1 (hydroxyxinnamic acids) and 2 (flavonoids) in all cases. This seems to indicate that there exists an important synergistic and/or additive action between the antioxidant compounds and other compounds present in the extract ,49. The 3+) to ferrous iron (Fe2+). The redox potential of the ferric tripyridyltriazine complex (0.70 V), which is involved in the FRAP reaction, is comparable to that of the ABTS●+ radical (0.68 V), and consequently, similar compounds will react in both ABTS and FRAP assays [The values of antioxidant capacity obtained with ABTS and FRAP assays were found within the same range, both for the total extract, as well as for the fractions rich in hydroxycinnamic acids and flavonoids. On the other hand, the antioxidant capacity obtained by means of the DPPH assay was lower in all cases. DPPH and ABTS methods are normally considered electron transfer-based assays, but hydrogen atom transfer also takes place in both methods, although in the DPPH assay, this mechanism is more marginal . The FRAP assays . FurtherP assays ,52, and P assays ,53. ThisP assays . The lowP assays  did not p < 0.01). This high correlation is not surprising, since the basic mechanism of all these methods is an oxidation/reduction reaction. Therefore, different authors recommend the use of TPC by the Folin–Ciocalteu assay to measure the antioxidant capacity of extracts as this is a simple method, which is reproducible, robust, and has a lot of comparable data in the literature [Folin–Ciocalteu reagent has been used for a long time to measure the total content of phenolic compounds in different samples, in spite of the fact that some non-phenolic compounds can also reduce this reactive, thus overestimating the TPC of the sample . Figure terature ,55.Considering that the highest antioxidant capacity and polyphenol content were found for the extracts obtained with methanol:water 60% v/v and ultrasounds, both fraction 1 and 2 were selected for the analysis of their antioxidant capacity on a model of the intestinal barrier. However, due to the insolubility of fraction 2 on cell culture medium, we were unable to evaluate its antioxidant capacity toward hydrogen peroxide insult on Caco-2 cells. Given the promising results obtained with FRAP, DPPH, and ABTS assays, fraction 2 could be a strong candidate for in vivo tests with animal models and further research will be carried out in this regard. Consequently, we analyzed the potential effect of fraction 1 on the protection of the intestinal barrier upon exogenous oxidative stress.The antioxidant capacity of fraction 1 (hydroxyxinnamic acids) was evaluated on Caco-2 cells. This cell line spontaneously acquires the phenotypic features of non-cancerous enterocytes after reaching confluence. Monolayer Caco-2 cells form tight junctions and present the cylindrical polarized morphology of enterocytes, expressing functional microvilli on the apical side ,57,58. T50 value of fraction 1 was calculated on Caco-2 cells in order to select a non-cytotoxic range of concentration. We obtained an IC50 value of 1023±159 μg/mL after long-term incubation (72 h). Therefore, we tested the antioxidant capacity of three concentrations below the obtained IC50 value  on the cells after 1 and 2 h of incubation, since such values did not display cytotoxic effects for short periods (data not shown). As can be observed in p < 0.05). This data encouraged us to determine the capacity of fraction 1 to reverse hydrogen peroxide insult on differentiated Caco-2 cells (2O2-induced ROS production (p < 0.05), whereas such behavior was not observed after 1 h incubation. Furthermore, the highest concentration of fraction 1 tested (1000 μg/mL) displayed a greater decrease in ROS levels when compared to the lowest one (250 μg/mL), which suggests the key role of extract concentration on its antioxidant capacity. Firstly, the IC-2 cells b. Simila® reduced oxidative stress biomarkers on pediatric patients of Crohn’s disease. Similarly, research performed on animal models of ulcerative colitis has revealed that the protective effect of plant extracts might be mediated by their antioxidant potential [The antioxidant capacity of plant extracts is strongly correlated with their clinical application for the management of oxidative stress-related gastrointestinal disorders. Koláček et al.  found thotential ,61. Our 2O2 exogenous addition. Ashwagandha, a traditional Indian herb, leaf extracts protected human neuroblastoma IMR32 cells from 2 h hydrogen peroxide insult after 24 h incubation with plant extracts at a concentration of 0.4 μg/mL [Calendula officinalis flowers prevented H2O2-induced damage, which might be interesting for the prevention of skin-related disorders, including melanoma [Sambucus nigra displayed a significant protective effect against various oxidative stresses on bovine aortic endothelial cells, and therefore might have a role in the prevention of cardiovascular disease [Hydrogen peroxide insult is a widespread method used to mimic the pro-oxidative environment that characterizes degenerative diseases such as cancer or neurodegenerative disorders on 2D cell cultures. In this line, plant-derived extracts have been investigated for their capacity to overcome the aberrant increase in ROS levels derived from H.4 μg/mL . Incubatmelanoma . Anthocy disease . Since, O-rutinoside, and luteolin-7-O-glucoside were the antioxidants present in a greater concentration in the mixture of by-products  from the “Blanca de Tudela” artichoke variety. Low amounts of cynarin, luteolin, apigenin-7-O-glucoside, apigenin-7-O-rutinoside, and naringenin-7-O-glucoside were also found. The extraction of hydroxycinnamic acids and flavonoids was much higher when 60% methanol was used as the extraction solvent in comparison to when water was used. In all cases, there was a good correlation between the concentration of total phenolics and the antioxidant capacity obtained by ABTS, DPPH, and FRAP. Of the three methods, FRAP is the only one that shows a good correlation with the concentration of the most abundant flavonoids in these extracts (luteolin derivatives), which would indicate that it is the best method for these kinds of samples. Our results also indicated that hydroxycinnamic acids from artichoke waste extracts might have a promising future in the management of oxidative stress on the gastrointestinal tract. Chlorogenic acid, luteolin-7-


This study indicates that the addition of OMMTs is a promising strategy to improve the properties of starch films.The knowledge gained from starch-nanocomposite-film research has not been fully applied commercially because of the lack of appropriate industrial processing techniques for nanofillers and starch films. Three organically modified montmorillonites (OMMTs) were prepared using a semidry kneading method. The effects of the OMMTs on the structures and properties of starch nanocomposite films, prepared by extrusion blowing, were investigated. The X-ray diffraction (XRD) analysis results revealed that the OMMTs with various quaternary ammonium salts possessed differing layer structures and d-space values. The results of the XRD and Fourier-transform infrared spectroscopy (FT-IR) showed that the starch–OMMT interaction resulted in a structural change, namely the starch–OMMT films possessed a balanced exfoliated and intercalated nanostructure, while the starch–MMT film possessed an exfoliated nanostructure with non-intercalated montmorillonite (MMT). The results of the solid-state nuclear magnetic resonance (NMR) analysis suggested that the starch-OMMT nanocomposite possessed comparatively large quantities of single-helix structures and micro-ordered amorphous regions. The starch–OMMT films exhibited good tensile strength (TS) (maximum of 6.09 MPa) and water barrier properties (minimum of 3.48 × 10 Traditional plastic packaging materials such as polyethylene, polypropylene, and polyvinyl chloride, which are derived from petroleum sources, are not biodegradable and cause serious ecological problems . To prevStarch is considered one of the most promising biodegradable substitutes for petrochemical plastics because of its abundance, low cost, renewability, and harmless degradation . HoweverOwing to its multilamellar structure, extremely large surface area, and intercalation properties, montmorillonite (MMT) has been used as an enhancer to improve the properties of bio-polyesters or starch plastics . In addi+ using three quaternary ammonium salts with various alkyl-chain lengths. The obtained OMMTs were then used to enhance starch films prepared by extrusion blowing. The multiple structural changes (owed to the introduction of the OMMTs occurring in the starch films) were investigated, in particular, the changes in the short-range- and micro-ordered regions. In addition, the changes in the mechanical properties and water vapor permeability (WVP) were investigated. The objective of this study is to reveal the mechanisms associated with various OMMTs, produced by a semidry kneading process, regarding their effects on the nanostructures and performances of starch films produced by extrusion blowing. In this work, OMMTs with various layer structures were prepared. This was achieved, using a semidry kneading method, by modifying MMT-Na+, with a cation exchange capacity (CEC) and d-spacing of 139.4 mmol/100 g and 1.47 nm, respectively, was purchased from Shouguang Zhonglian Fine Montmorillonite Co., Ltd. . The surfactants that were used to modify MMT-Na+ were purchased from Shandong Long Chain Chemical Co., Ltd. . Anhydrous ethanol and glycerol were purchased from Tianjin Kaitong Chemical Reagent Co., Ltd. .Hydroxypropyl di-starch phosphate (HPDSP) was purchased from Hangzhou Starpro Co., Ltd. . MMT-Na+ was modified using three quaternary ammonium salts, via a semidry kneading method. Quaternary ammonium salt (750 g) was dissolved in anhydrous ethanol (400 g) in a water bath at 80 °C. Then, the surfactant solution was mixed with the MMT-Na+ (3 kg) that had been preheated at 80 °C in a kneading machine. The mud-like blend was kneaded at 80 °C for 60 min in a sealed kneading machine. This was then continued for another 30 min using an open kneading machine at the same temperature. Subsequently, the OMMT was washed in hot aqueous ethanol and dried in an oven for 12 h. Finally, it was ground and sifted through a 200-mesh sieve. The OMMTs containing modifiers 1231, 1631, and 1831 were denoted as 1231-MMT, 1631-MMT, and 1831-MMT, respectively. The structures of the quaternary ammonium salts, as well as the CEC values and d-spacings of the OMMTs determined according to the method of Gao et al. [MMT-Naw/w) MMT-Na+ or OMMT were blended in an SHR50A mixer  for 5 min at 500 rpm. Then, 30%  glycerol was added, and the mixture was blended for 10 min at 1500 rpm. The samples containing MMT-Na+, 1231-MMT, 1631-MMT, and 1831-MMT were denoted as HPDSP-Na, HPDSP-1231, HPDSP-1631, and HPDSP-1831, respectively. The mixtures were sealed in polyethylene bags for 24 h at room temperature to equilibrate the components. HPDSP and 10% , with a screw diameter of 35 mm and screw length of 30 D; temperatures of 80 °C and 120 °C were used in zones I and II of the barrel, respectively, and a screw speed of 20 rpm was employed. The HPDSP nanocomposite films were blown using a single-screw extruder with a screw diameter of 35 mm, screw length of 25 D, screw compression ratio of 3:1, and five individually controlled temperature zones. The extruder was equipped with a conventional temperature-controlled film-blowing die with a diameter of 60 mm and a film-blowing tower with a calendering nip and takeoff rolls . The temperatures between the feed inlet and the die were 80 °C, 120 °C, 130 °C, 135 °C, and 125 °C. The ratio of the diameter of the blown bubble to that of the die (blow-up ratio) was carefully adjusted to 3:1. At the die exit, the ratio of the take-up velocity to the film velocity (take-up ratio) was carefully adjusted to 4:1. Prior to testing, all the films were stored for at least seven days within a chamber maintained at a constant temperature of 23 °C and relative humidity (RH) of 53% .The thickness of the HPDSP nanocomposite films was determined using 211-101F micrometers . Each film was tested randomly for six times and the thickness of the film was obtained by averaging the results of the six replicates. The mechanical properties of the HPDSP nanocomposite films were determined, according to the ASTM-D882-02 standard test method (2002), using a TA-XT2i texture analyzer . Each film was cut into five strips, each with a length and diameter of 90 and 15 mm, respectively. The initial distance between the grips was 50 mm, and the test speed was 60 mm/min. The tensile strength  and elongation at break  values of the films were obtained by averaging the results of the six replicates. The WVP was determined using a PERMETMW3/030 water vapor permeability tester . The films were cut into round specimens (80 mm in diameter) using a special sampler. The WVP was determined at a temperature and RH of 38.0 °C and 90%, respectively, using a preheating time of 4 h and weighing interval of 120 min. The WVP of each sample was obtained by averaging the results of the three separate tests. +, OMMTs, and HPDSP nanocomposite films. This was performed using a D8 Advance X-ray diffractometer  equipped with a copper target, λ = 0.15406 nm; the radiation parameters were 40 kV and 30 mA, and a slit of 2 mm was used. In the case of the MMT-Na+ and OMMT films, XRD patterns were acquired over the 2θ range of 1 to 10°, while a range of 1–30° was used for the HPDSP nanocomposite films. A step size and step rate of 0.02° and 0.5 s were used, respectively. X-ray diffraction (XRD) was conducted to analyze the MMT-Na−1, respectively. All spectra were baseline corrected over the range of 1200–900 cm−1. The line shape was assumed to be Lorentzian, with a resolution enhancement factor and half-width of 1.9 and 19 cm−1, respectively. The infrared absorbance values at 1047 and 1022 cm−1 were extracted from the spectra following baseline correction and normalization of the second derivative spectra using OMNIC software. Then, the ratio of the absorbance at 1047/1022cm−1 was determined. Fourier-transform infrared (FT-IR) analysis was performed on the HPDSP nanocomposite films, using a Nicolet iS 5 FT-IR analyzers with an attenuated total reflectance (ATR) detector . The number of accumulated scans and the scanning rate were 32 and 4 cm13C NMR) was performed using a Bruker Avance III 400 MHz (1H/proton resonance) Wide Bore  equipped with a 4-mm broadband double-resonance CP/MAS probe. Each film sample (500 mg) was placed into the rotor and introduced to the center of a magnetic field. The spectra were decomposed using PeakFit software version 4.0 . Solid-state nuclear magnetic resonance . This was followed by post-hoc Duncan’s multiple range tests using SPSS software 21 . + and OMMTs modified by the various quaternary ammonium salts are shown in + can be observed at 2θ = 5.98°, which is equivalent to a basal spacing of 1.47 nm. In the case of the OMMTs modified with quaternary ammonium salts, the characteristic peaks shift to lower angles. This indicates that surfactants 1231, 1631, and 1831 were intercalated into the MMT layers via the semidry kneading method. This resulted in the expansion of the d-spacings from 1.47 nm (MMT-Na+) to 1.76, 2.02, and 2.06 nm, respectively . By comparing the intensities and shapes of the peaks, it can be concluded that amongst the OMMTs, 1631-MMT had the most ordered layer structure, and 1231-MMT had the most disordered structure [The XRD patterns of MMT-Naectively . These rtructure . + and OMMTs within the HPDSP matrix was determined by measuring the characteristic peaks within the XRD patterns , C4 (81.4 ppm), C2,3,5 (72.2 ppm), and C6 (61.9 ppm) correspond to the carbon atoms within the HPDSP molecules, as shown in The  helices . As show carbons A. The pe1 peak (100–103 ppm) can be resolved into the double-helix (series of peaks near 100 ppm) and single-helix (103 ppm) components of the starch molecule [1 peaks in the spectra of the HPDSP-Na film, a broad peak was observed near 103 ppm, and a series of shoulder peaks were observed near 100 ppm. These observations suggest that the starch molecules of the nanocomposites primarily consist of a single-helix V-type structure with a number of double helices. Following the addition of OMMT to the starch film, the area of the peak at 103 ppm increased (+. This promoted the double-helix structure to transform into a single-helix structure. A sharper C1 peak was observed in the spectra of the HPDSP-1631 film, which suggests that the 1631-MMT contributes to homogeneous distribution by interrupting inter- and intramolecular hydrogen bonds [The ordered spectrum at the Cmolecule . Considencreased , and thencreased A. This ien bonds . In addien bonds .4 peak is considered characteristic of the amorphous glassy phase of starch [4 peak area. This suggests that further amorphization of the starch film occurred. The total change in the short-order structure of the starch film, prepared by extrusion blowing and with various OMMTs, was calculated using Liu’s method [The Cf starch . When vas method  (Table 3s method . One reas method , as indi+, the hydrophobic surface and exfoliated layers of the OMMTs enhanced the water vapor barrier. Various OMMTs influenced the proprieties of the starch film by changing the balance between the exfoliated and intercalated starch-MMT layer systems and the short-range structures of the amorphous and crystalline regions. The starch film with 1631-MMT had more single helix structures and micro-ordered amorphous regions, making it more suitable than 1231-MMT and 1831-MMT for enhancing HPDSP films prepared by extrusion blowing. The HPDSP nanocomposite film is a promising material for food inner packaging due to its good tensile strength and barrier properties. The effects of OMMTs, prepared by a semidry kneading process, on the properties of starch films, formed by extrusion blowing, were investigated. Following the addition of the OMMTs, the starch films exhibited higher TS values and improved EAB values. This was owing to the formation of a greater quantity of micro-ordered regions with single-helix structures, which prevented changes in the molecular orientation. Compared with the results obtained for the film containing MMT-Na


Ms) of TNT-CoHA 12.6 times higher than that of Ti-CoHA. The contrast in MRIs is related to the magnetic properties of the particles. Therefore, TNT-CoHA has good image contrast at low concentrations in T2 images. The relaxivity coefficient of the CoHA was higher for TNT-CoHA (340.3 mM−1s−1) than Ti-CoHA (211.7 mM−1s−1), and both were higher than the commercial iron nanoparticles (103.0 mM−1s−1). We showed that the TNT substrate caused an increase in the size of the cobalt oxide crystal of TNT-CoHA, thus effectively improving the magnetic field strength and MRI image recognition. It was also shown that the relaxivity coefficient rose with the Ms. Evaluation of biocompatibility of CoHA using human osteosarcoma cells (MG63) indicated no toxic effects. On the other hand, CoHA had an excellent antibacterial effect, as shown by E. coli evaluation, and the effect of TNT-CoHA powder was higher than that of Ti-CoHA powder. In summary, TNT-CoHA deposited electrochemically on the TNT substrates can be considered as a potential candidate for the application as an MRI contrast agent. This paper is a comparative study of how different electrode plates affect the magnetic and MRI image contrast of cobalt-substituted hydroxyapatite (CoHA) nanomaterials.Magnetic nanoparticles are used to enhance the image contrast of magnetic resonance imaging (MRI). However, the development of magnetic nanoparticles with a low dose/high image contrast and non-toxicity is currently a major challenge. In this study, cobalt-substituted hydroxyapatite nanoparticles deposited on titanium (Ti-CoHA) and cobalt-substituted hydroxyapatite nanoparticles deposited on titanium dioxide nanotubes (TNT-CoHA) were synthesized by the electrochemical deposition method. The particle sizes of Ti-CoHA and TNT-CoHA were 418.6 nm and 127.5 nm, respectively, as observed using FE-SEM. It was shown that CoHA can be obtained with a smaller particle size using a titanium dioxide nanotube (TNT) electrode plate. However, the particle size of TNT-CoHA is smaller than that of Ti-CoHA. The crystal size of the internal cobalt oxide of CoHA was calculated by using an XRD pattern. The results indicate that the crystal size of cobalt oxide in TNT-CoHA is larger than that of the cobalt oxide in Ti-CoHA. The larger crystal size of the cobalt oxide in TNT-CoHA makes the saturation magnetization ( The surt pH 6.5 . The prer system . The E. 2PO4, 8 g NaCl, and 2.16 g Na2HPO4.One tenth of a gram each of Ti-CoHA and TNT-CoHA were immersed in 10 mL of phosphate buffered saline (PBS), placed in a constant temperature water bath (37 °C) for four weeks, and the pH was measured daily. The PBS composition was 0.2 g KCL, 0.2 g KH®14.1.0, Cary, NC, USA). One-way ANOVA was used to examine the differences between groups using the Tukey HSD multiple comparisons. A value of p < 0.05 was considered to be significant. All data are from the average of three repetitions ± standard deviation. Data were calculated using JMP14 software (JMPThe pure titanium test piece was anodized to obtain a flat TNT B. The SE2 (ICSD. No-024080) at 28.9° and 30.0° [−1 and 1053 cm−1 represent −1 represents −1 and 3438 cm−1 represent −OH [The appearance of the Ti-CoHA and TNT-CoHA powders was observed to be spherical by SEM A–D. FE-Snd 30.0° . TNT-CoHnd 30.0° . The chaHc), saturation magnetization (Ms), and remnant magnetization (Mr) of Ti-CoHA and TNT-CoHA are summarized as Mr/Ms) is an important characteristic parameter for magnetic material applications. The literature indicates that the squareness ratio of a material will be reduced due to the decrease in Co content [Mr/Ms of the two samples is less than 0.5, indicating that the particles interact through magnetostatic interactions [Ms and Mr of TNT-CoHA are 12.6 times and 13.5 times larger than those of Ti-CoHA, respectively. Magnetic properties depend on the magnetization direction, morphology, and crystallinity of the material [Ms. However, the increasing particle size may be detrimental to pharmacokinetics and biodistribution, so the development of small magnetic nanoparticles with high magnetic saturation is the main target [Being superparamagnetic is an important condition for nanoparticles to possess. Superparamagnetic nanoparticles can be applied to stem cell therapy, drug delivery, and contrast agents.  content . Thereforactions . It is wmaterial . V. Saramaterial . Those rmaterial ,37. Sincmaterial , were apn target . We used2WI magnetic resonance imaging, reducing the intensity of tissue signals in the image and increasing the contrast of the surrounding image [2WI imaging with increasing concentration, but Ti-CoHA and TNT-CoHA improved the image contrast (2WI MRI (2 (R2 = 1/T2) is usually used to indicate the paramagnetic effect. The higher the R2, the shorter the T2 relaxation time, the darker the tissue image, and the larger the contrast effect compared with the tissue with lower R2. In addition, the R2 value of TNT-CoHA (R2 = 46) was superior to that of Ti-CoHA (R2 = 33) at a cobalt ion concentration of 0.12 Mm  obtained from the gradient of the R2 curve and the magnetic nanoparticle molar concentration is a standardized contrast intensity index [2 value, the lower the concentration of nanoparticles required in the same T2 image. This avoids excessive metal ion accumulation. The calculated relaxivity coefficient of the CoHA magnetic nanoparticles was higher for TNT-CoHA (340.3 mM−1s−1) than Ti-CoHA (211.7 mM−1s−1). However, there are many factors affecting the relaxation efficiency coefficient such as particle size, crystal size, surface effect, and saturation magnetic field strength [2WI imaging and thus increases the image contrast. Another plausible reason is attributed to the fact that the Ms of TNT-CoHA is higher than that of Ti-CoHA. We put together a comparison of the magnetic nanoparticle relaxivity coefficients reported in the literature  with commercial iron nanoparticles Endorem® NP  as an MRI contrast agent in the liver [2 values of FeHA and Endorem® NP were 165 mM−1s−1 and 103 mM−1s−1, respectively, and both showed a good contrast. This study and the aforementioned FeHA both used ion-substituted HA. Since the r2 values are higher in this study than in [2WI contrast agents. Furthermore, the magnetic nanoparticle size clinically approved for use in contrast agents is 10–300 nm [The magnetic dipole moment is induced in superparamagnetic nanoparticles under an applied magnetic field. This change results in the magnetic nanoparticles being a negative contrast in Tng image . Commerccontrast A. The Co. The quantitative results of calcium deposition by Alizarin Red S (ARS) staining are shown in p ˂ 0.05) after 3 and 7 days of cell culture. The results showed that the two groups of CoHA were not toxic and TNT-CoHA contributed to cell growth. In addition, bacterial infections around the wound are an important challenge for bone repair materials. Previous studies have reported that Co3O4 effectively inhibits the growth of E. coli and can be used as an antibacterial drug [E. coli to evaluate the antibacterial properties of CoHA by colony counting. The number of colonies in the TNT-CoHA was significantly lower than that of the other groups after 24 h of culture . Residues of the contrast agent and target in the body may have a negative impact on the human body. Therefore, CoHA was immersed in PBS to simulate changes in the pH of the human body  was used as a control group. MTT results showed no significant differences in initial cell attachment A. The suial drug . Accordi culture C. Quantiman body D. The re2 image shows that TNT-CoHA has good image contrast at low concentrations, avoiding the accumulation of high doses of magnetic nanoparticles in the body. The relaxivity coefficient of the sample showed that TNT-CoHA (340.3 mM−1s−1) was higher than that of Ti-CoHA (211.7 mM−1s−1), and both were higher than that of the commercial iron nanoparticles (103.0 mM−1s−1). In addition, Ti-CoHA and TNT-CoHA are not cytotoxic. In short, the use of titanium dioxide nanotubes as a substrate effectively improves the saturation magnetic field strength and magnetic resonance contrast of CoHA and has good biocompatibility and antibacterial properties. These results indicate that TNT-CoHA might be effectively used as an MRI contrast agent or targeting material in the future and may be helpful for the development of medical imaging materials.In this study, we synthesized Ti-CoHA and TNT-CoHA magnetic nanoparticles by electrochemical deposition using two different substrates. It was found that the crystal size of the cobalt oxide of the nanoparticle strongly influenced the degree of magnetization and the relaxivity coefficient. Although the particle size of TNT-CoHA is smaller than that of Ti-CoHA, the internal cobalt oxide of TNT-CoHA has a larger crystal size than Ti-CoHA, thus, TNT-CoHA has a saturation magnetic field strength of up 12.6 times that of Ti-CoHA. The T


We report here a novel electrochemical sensor developed using fluorine-doped graphene oxide (F-GO) for the detection of caffeic acid (CA). The synthesized graphene oxide (GO) and F-GO nanomaterials were systematically characterized with a scanning electron microscope (SEM), and the presence of semi-ionic bonds was confirmed in the F-GO using X-ray photoelectron spectroscopy. The electrochemical behaviours of bare glassy carbon electrode (GCE), F-GO/GCE, and GO/GCE toward the oxidation of CA were studied using cyclic voltammetry (CV), and the results obtained from the CV investigation revealed that F-GO/GCE exhibited the highest electrochemically active surface area and electrocatalytic activity in contrast to the other electrodes. Differential pulse voltammetry (DPV) was employed for the analytical quantitation of CA, and the F-GO/GCE produced a stable oxidation signal over the selected CA concentration range (0.5 to 100.0 μM) with a low limit of detection of 0.018 μM. Furthermore, the acquired results from the selectivity studies revealed a strong anti-interference capability of the F-GO/GCE in the presence of other hydroxycinnamic acids and ascorbic acid. Moreover, the F-GO/GCE offered a good sensitivity, long-term stability, and an excellent reproducibility. The practical application of the electrochemical F-GO sensor was verified using various brands of commercially available wine. The developed electrochemical sensor successfully displayed its ability to directly detect CA in wine samples without pretreatment, making it a promising candidate for food and beverage quality control. Therefore, the development of sensitive and selective electrochemical sensors for the quantitative detection of CA is essential.Caffeic acid  is an important compound in the classification of phenolic acids, which exists in certain vegetables  and fruits . It serves as an antioxidant, anti-inflammatory, antibacterial, and immune-modulating agent ,2,3,4. TSeveral previously reported works have focused on the quantitative determination of CA utilizing different types of nanostructured materials in various electrolyte media ,16,17,18The doping of graphene and GO with heteroatoms  at the molecular and atomic levels favourably enhances their electrochemical behaviour and electrocatalytic properties while altering their electronic and physicochemical properties ,44,45. BThe aims of the present study were two-fold: (i) to compare the electrochemical properties of GO and F-GO as an advanced electrochemical sensing material and (ii) to develop a high-performance electrochemical sensor for the sensitive detection of CA. To the best of our knowledge, this is the first report on a highly sensitive and selective F-GO-based electrochemical sensor for the direct measurement of CA in different brands of wine.® diamond™ UV water purification system), and all glassware was thoroughly washed and dried prior to each analysis. Four different brands of red wine were purchased from a liquor retail store. All experiments were conducted using a Britton–Robinson (B-R) electrolyte medium 3−/4− in 0.1 M KCl). 6]3−/4− at the bare GCE, GO/GCE, and F-GO/GCE. In contrast to the bare GCE and GO/GCE, the F-GO/GCE demonstrated a higher anodic-cathodic peak current and a smaller peak-to-peak potential separation. The potential separations for the bare GCE, GO/GCE, and F-GO/GCE were calculated to be 294, 158, and 113 mV, respectively. The smaller peak-to-peak separation demonstrated a better electrochemical reversibility and an improved electron transfer efficiency 3−/4− redox probe at the F-GO/GCE was reversible. Subsequently, −1) in the KCl-ferricyanide medium to study the redox behaviour of the modified electrode and to calculate the electrochemically active surface area (EASA) of the F-GO/GCE using the Randles–Sevcik equation 3−/4− molecules in mol L−1, A is the EASA (cm2), n signifies the electron transfer number, and ʋ is the scan rate (V s−1). 1/2 plots; the EASA was calculated to be 0.045, 0.075, and 0.745 cm2 for the bare GCE, GO/GCE, and F-GO/GCE, respectively. These observed results verified that the fabricated F-GO/GCE had a higher EASA compared to GO and bare GCE.Furthermore, the value obtained from the ratio of the anodic to cathodic peak current (Ipa/Ipc = 0.96) confirmed that the oxidation-reduction of the [Fe(CN)equation :(1)ip=2−1 in the absence of CA. There were no anodic/cathodic peaks that appeared for the prepared GCEs without CA in the B-R buffer solution. It was evident from the CV that the F-GO/GCE had a larger background current when compared to the bare GCE and GO/GCE, which further confirmed that F-GO had a much larger EASA.Cyclic voltammetry was primarily employed to investigate the electrooxidation behavior of CA at the F-GO modified GCE. From Ep values calculated from the CA oxidation/reduction peak potentials with F-GO/GCE (90 mV) were lower than that of the bare GCE (117 mV), further confirming that F-GO/GCE possessed a good electrocatalytic activity toward the oxidation of CA.The F-GO/GCE amplified the rate of the electron transfer and enhanced the overall response toward the electrochemical oxidation of CA. The current response recorded with the F-GO/GCE showed a higher anodic and cathodic peak intensity for the oxidation/reduction of CA as opposed to the bare GCE and GO/GCE. The current response of the F-GO/GCE was 1.5 and 50 times higher than that of the GO/GCE and the bare GCE, respectively. The presence of the C–F semi-ionic bond and a higher conductivity facilitated the higher electrocatalytic oxidation of CA. Moreover, the Δ−1. The values of the anodic/cathodic peak current were incremental with the increased scan rates; however, the observations from the CV exhibited a shift in the oxidation/reduction peak potentials, indicating that the electrochemical oxidation/reduction process was quasi-reversible. At higher scan rates, the electrochemical reaction became less reversible due to the slow electron-transfer rate [Based on the effect of the scan rate, the electrocatalytic oxidation of CA was investigated further by employing CV. fer rate ,67.−1) is presented in 2 values and the linear regression equation from the calibration plot were R2 = 0.9960, Ipa = 1.292 × 10−6 A/mV s−1 + 15.119 µA and R2 = 0.9921, Ipc = −1.114 × 10−6 A/mV s−1 − 17.114 µA , which contained 30.0 µM of the analyte at a scan rate of 50 mV s−1 under different concentrations. It is evident from 2 values of 0.9942 for Ipa and 0.9951 for Ipc.The oxidation behavior of CA was further investigated in a 0.1 M B-R buffer solution (pH = 2.65) at a scan rate of 50 mV sThe analytical capacity of the F-GO/GCE was investigated by employing DPV. In contrast to CV, DPV is more sensitive and has the ability to lower the background current . Figure 2 coefficient values from the calibration plot were calculated to be 0.9960 and 0.9971, respectively. The sensitivities from the calibration plots were calculated to be 5.27 and 0.35 µAµM−1 cm−2. The limit of detection (LOD) was further calculated to be 0.018 µM using the formula 3 σ/s, where σ denotes the standard deviation of three blank measurements and s represents the slope calculated from the calibration plot. The two calibration plots acquired in this study were similar to studies that involved the measurement of molecules, such as acetaminophen and valacyclovir, ascorbic acid, dopamine, uric acid, and NADH [The linear regression equation between the concentration  and the current  for the electrochemical oxidation of CA in the concentration ranges from 0.5–10.0 μM and 15.0–100.0 μM B were Ipand NADH ,59,71,72and NADH ,73. ImpoThe stability of the sensor was scrutinized by employing DPV for 30 consecutive scans A. The sep-coumaric acid, hydroquinone, trans-ferulic acid, gallic acid, glucose, and ascorbic acid. The results obtained from the interference study revealed that the F-GO/GCE sensor retained 92% of its activity for 100.0 µM CA oxidation in the presence of interference molecules. Therefore, the developed F-GO/GCE electrochemical sensor could be applied to the real-time analysis of CA.The selectivity of the developed electrochemical sensor was an essential factor for the detection of CA to enable the testing of actual samples. Henceforth, the prepared F-GO/GCE sensor was tested for its anti-interference capabilities in the presence of potentially interfering species using DPV. The electrochemical performance of the designed F-GO/GCE sensor was further validated for its real-time determination of CA in wine. Four different samples of red wine were used for the determination of CA. The wine was tested by injecting raw samples directly into the 0.1 M B-R buffer solution, and the concentrations of CA were determined from their respective DPV peaks. The DPV peaks obtained for the determination of CA in wine samples (50.0 µL to 300.0 µL) are included in the The exact procedure was followed for analyzing the concentrations of CA in other wine samples, and the results of the quantification of CA in the assorted brands of wine are presented in A highly selective F-GO based sensor was developed for the sensitive detection of CA in assorted brands of wine. The results obtained from the CV investigations indicated that the F-GOs enhanced the electrocatalytic behaviour in contrast to bare GCE and GO modified GCE. The F-GO/GCE exhibited a higher EASA and improved the electrochemical performance as opposed to the GO/GCE. The analytical quantitation performed using DPV revealed the strong capability of the electrochemical sensor for the determination of CA within a wide linear concentration range in a 0.1 M B-R buffer (pH 2.65). The F-GO/GCE sensor demonstrated a superior selectivity for the oxidation of CA in the presence of additional phenolic compounds. The developed electrochemical sensor exhibited a good long-term stability and sensitivity, a low detection limit, and a satisfactory reproducibility. Further, it was effectively employed for the detection and quantification of CA in various brands of red wine. The sensor also displayed a superior sensing capacity to detect CA directly in wine without the requirement of an external electrolyte medium. These collective factors confirmed that the developed F-GO/GCE sensor could be incorporated for beverage quality control.


Tumorous metastasis is a difficult challenge to resolve for researchers and for clinicians. Targeted delivery of antitumor drugs towards tumor cells’ nuclei can be a practical approach to resolving this issue. This work describes an efficient nuclear-targeting delivery system prepared from trans-activating transcriptional activator (TAT) peptide-functionalized graphene nanocarriers. The TAT peptide, originally observed in a human immunodeficiency virus 1 (HIV-1), was incorporated with graphene via an edge-functionalized ball-milling method developed by the author’s research group. High tumor-targeting capability of the resulting nanocarrier was realized by the strong affinity between TAT and the nuclei of cancer cells, along with the enhanced permeability and retention (EPR) effect of two-dimensional graphene nanosheets. Subsequently, a common antitumor drug, mitomycin C (MMC), was covalently linked to the TAT-functionalized graphene (TG) to form a nuclear-targeted nanodrug MMC-TG. The presence of nanomaterials inside the nuclei of ocular choroidal melanoma (OCM-1) cells was shown using transmission electron microscopy (TEM) and confocal laser scanning microscopy. In vitro results from a Transwell co-culture system showed that most of the MMC-TG nanodrugs were delivered in a targeted manner to the tumorous OCM-1 cells, while a very small amount of MMC-TG was delivered in a non-targeted manner to normal human retinal pigment epithelial (ARPE-19) cells. TEM results further confirmed that apoptosis of OCM-1 cells was started from the lysis of nuclear substances, followed by the disappearance of nuclear membrane and cytoplasm. This suggests that the as-synthesized MMC-TG is a promising nuclear-target nanodrugfor resolution of tumorous metastasis issues at the headstream. New drug delivery systems are required thatare highly active in the body and have a high specific targeting capability. This goal is considered an ultimate outcome for chemotherapy. For example, the biggest challenge for the clinic utilization of ophthalmic drugs is their low bioavailability as the blood retina and the cornea act as barriers . Novel nChoroidal melanoma (CM) is a primary ocular malignant tumor observed frequently amongst adults. Although numerous techniques, including resection, enucleation, radiotherapy, and laser therapy, can be used to treat CM, significant side effects from these treatments are inevitable ,9. For eIn this work, a novel nanoscale drug delivery system was formulated and had a high nuclear-targeting capacity. This property was observed after combining the advantages of the unique size of graphene materials with the properties of trans-activating transcriptional activator (TAT). Graphene-based nanomaterials (particularly those that are less than 200 nm in diameter) can pass through the leaky tumor vessels and can accumulate in the tumor and in its microenvironment. This is due to the enhanced permeability and retention (EPR) effect of nanomaterials ,14. In tIn the present work, nanosheets from TAT-functionalized graphene (TG) were prepared via an efficient edge-functionalized ball-milling (EFBM) method as reported by the author’s research group previously ,18. TAT −1 , 1580 cm−1 , and 2700 cm−1 (2D band). Compared to the spectrum of pristine graphite  spectroscopy was used to confirm the immobilization of MMC into TG. As seen in The drug-loading and encapsulation efficiencies of the MMC-TG were found to be 22% and 55%, respectively, and were calculated using a standard curve of the composite’s UV absorption intensity measured at 363 nm for different MMC concentrations . The in −1 (N–H stretching vibration), 1640 cm−1 (C=O stretching vibration), 1580 cm−1 (N–H bending vibration), and 1370 cm−1 (C–N stretching vibration). This supports the successful covalent linkage of MMC to TG. Characteristic peaks of MMC were seen in the spectrum of MMC-TG at 2960 cm−1 (C–H stretching vibration of aromatic rings) and in the range of 1500 to 1600 cm−1 (C=C stretching vibration of aromatic rings). This suggests the presence of MMC in the resulting MMC-TG.Further evidence confirming the successful synthesis of MMC-TG was obtained from the Fourier transform infrared (FTIR) spectra . The FTITo test the targeted antitumor capability of the as-synthesized MMC-TG, a Transwell two-cell co-culture system was used . TypicalThe cell viability of OCM-1 was further reduced to 5% when the cells were incubated with MMC-TG and consisted of 4 µg/mL MMC for 72 h, suggesting the strong tumor lethality of the MMC-TG nanodrugs. On the other hand, it was found that the pristine MMC induced indistinctive toxicity in both cell types b. Lower Based on these cell viability results, samples containing 4 µg/mL MMC were tested with a calcein acetoxymethyl ester  staining assay. In this assay, the live cells appeared green after cells were stained by the fluorescent dye Calcein-AM. As shown in TM-X phalloidin . As shown in To better understand the distribution of TG inside OCM-1 cells, we imaged the OCM-1 cells using a confocal laser scanning microscope (CLSM) labeled with fluorescein isothiocyanate (FITC)-phalloidin and 4′,6-diamidino-2-phenylindole (DAPI). As shown in Flow cytometry was further also applied to the quantitative analysis of TG distribution inside OCM-1 cells. TG stained with FITC was incubated with OCM-1 cells for 24, 48, and 72 h. The as-prepared cells were collected and measured with flow cytometry. As shown in In addition, we carried out TEM characterization on OCM-l cells cultured with MMC-TG to further confirm the nuclear-targeting capability of the nanodrugs. The eye is an essential organ of a human body. More than 80% of our external information is obtained via our eyes . Hence, In this work, TAT, a type of cationic cell-penetrating peptide, was used to better target the nuclei of cancer cells. In particular, its ability to penetrate the cell membrane and its nuclear membrane was tested. In order to optimize the targeting and drug-loading impact, two-dimensional graphene nanosheets were bound with TAT via an EFBM procedure developed by the author’s research group. This method provided an environmentally friendly and efficient procedure to prepare TAT-functionalized graphene (TG) that had minimal structural defects. No chemical redox reaction was involved during its fabrication. A common antitumor drug, MMC, was then introduced into the TG nanosized drug-carrier. To the best of our knowledge, MMC has not been reported elsewhere before for the treatment of uveal melanoma including CM. This may be due to the myelosuppression issues related to MMC administration and to the inconvenience of administrating fundus . Some stThe resulting MMC-TG nanodrug had more than a 95% specific tumor suppression rate and, at the same time, a low toxicity towards normal cells. This was confirmed by both a Transwell two-cell co-culture system and Calcein-AM live cell-staining tests. Its high impact on tumor inhibition was attributed to the outstanding targeted nuclear penetration capability of TG. CLSM and TEM results confirmed the presence of TG-based nanomaterials inside cancer cell nuclei. This suggests that the TG nanocarrier was able to carry the MMC drug into the nuclei of tumor cells. The TEM results indicate that the apoptosis of tumor cells was caused by the lysis of substances within the cell’s nuclei and confirm that most MMC-TGs were accumulated inside the nuclei first. Thus, the damage of nuclear substances induced by the nanodrug occurs at this initial stage. Since the cell nucleus is the source of gene inheritance and transcription, it has not escaped our notice that preventing tumor cells from initiating metastasis, as seen in this proposed treatment, is a very useful property. The as-synthesized novel nuclear-targeting drug delivery system reported here promises the great possibility of resolving the tumorous metastasis challenge that has troubled both fundamental researchers and clinical doctors treating this usually fatal disease. The myelosuppression issue of MMC and the biosafety problem of graphene in other human body systems have yet to be settled. Thus, the best administration routine for clinical utilization of MMC-TG, in our opinion, is via topical administration.Graphite flakes were kindly provided by Haida Corporation . TAT was purchased from GL Biochem Ltd. . 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC• HCl), N-hydroxylsuccinimide (NHS), and Mitomycin C (MMC) were obtained from Sigma-Aldrich . Transwell plates were supplied by Corning Incorporated. The CCK-8 was sourced from Dojindo Company . OCM-1 and ARPE-19 cells were purchased from the American Type Culture Collection (ATCC) . DMEM/F12 culture medium, 1640 culture medium, Fetal Bovine Serum (FBS), and trypsin were obtained from Gibco .The physiochemical properties of nanomaterials were characterized using Raman spectroscopy , FTIR , and UV-vis . The morphologies of nanomaterials were measured using an AFM  in a tapping mode.TG was synthesized via a self-discovered, one-step EFBM method at room temperature. Typically, 10 mg of TAT and 10 mg of graphite flakes were added to a 400-mL milling capsule. These mixes were vigorously shaken at the speed of 400 rpm for 4 h in a planetary ball-milling machine . Twenty mL of deionized (DI) water was then used to remove the milled mixture from the capsule. The samples were subsequently centrifuged at 1000 rpm for 10 min to remove unreacted graphite in the sediment. The supernatant was further treated by centrifuging at 8000 rpm for 10 min. This step was repeated 3 more times to remove any unreacted TAT. The thus treated TG was dispersed in deionized water for further testing. To prepare the TG powder, the as-synthesized TG dispersion was freeze-dried for 24 h. The as-prepared nanomaterial powder was treated with plasma in acetic acid vapor at 50 W for 5 min to introduce carboxyl groups to the TG surface so that the MMC could be immobilized. TG was then soaked in the aqueous solution containing NHS and EDC for 2 h to activate its carboxyl groups. MMC was then covalently loaded to TG via an amidation reaction run at 4 °C for 48 h. The formed MMC-TG was gathered by centrifuging at 8000 rpm for 10 min. This step removed residual NHS, EDC, and unreacted MMC.The Transwell system consisted of a microporous membrane between two compartments. This was used to co-culture two kinds of cells in the same culture medium simultaneously. In this work, normal ARPE-19 cells were seeded in the upper chamber of the Transwell plate and tumorous OCM-1 cells were cultured in the lower compartment. MMC and MMC-TG dispersed in culture medium were added to the Transwell system at MMC densities of 1 µg/mL, 2 µg/mL, 3 µg/mL, and 4 µg/mL after the cells were attached to each compartment. The cell viability was evaluated using the cell counting kit-8 (CCK-8) for different incubation periods. The live staining of cells were performed using a calcein acetoxymethyl ester  stain kit, followed by its fluorescence microscopy characterization.TG was incubated with OCM-1 for 6 to 72 h. Cells were fixed in 4% paraformaldehyde for 15 min, followed by permeabilizing with 0.1% Tritonx-100 for 5 min. Cell cytoplasm was stained with fluorescein isothiocyanate (FITC) labeled with phalloidin. The cell nucleus was stained with 4,6-diamidino-2-phenylindole (DAPI) for 5 min. Distribution of nanomaterials inside cells was identified using a confocal laser scanning microscope .3 cells/well. The plates were placed in a cell culture incubator for 24 h to allow the cell to spread. Four µg/mL of TG stained with FITC was then co-cultured with OCM-1 cells for various time periods. The cells were harvested at 24, 48, and 72 h. Cells were washed three times with PBS before measurement with the flow cytometry.Flow cytometry measured the percentage of OCM-1 cells containing TG. Measurement was based on the fluorescence intensity of the cells. OCM-1 cells were seeded in 6-well culture plates at a cell density of 20 × 10OCM-1 cells were pre-cultured with 4 µg/mL MMC-TG for 24 and 72 h. Cells were collected and prefixed with 2.5% glutaraldehyde at 4 °C for 15 min. Cells were then postfixed with 1% osmic acid at 37 °C for 1 h, followed by staining with 1% uranyl acetate at 37 °C for 1 h. Cells were gradient dehydrated with different acetone solutions at various concentrations and then soaked in embedding liquid at 37 °C overnight. The cells were then embedded in a polymerizing liquid to allow them to be solidified. The cell blocks were sliced to ultrathin sections for their characterization with TEM .In summary, we successfully prepared a highly efficient nuclear-targeting antitumor drug-carrier formed from MMC-immobilized TG. The strong nuclear-targeting capability was achieved by the incorporation of TAT with two-dimensional nanosheet graphene using an environmentally friendly edge-functionalized ball-milling method. MMC was covalently immobilized onto TG. The as-synthesized MMC-TG had a strong suppression capability (with a more than 95% suppression efficacy) towards tumorous OCM-1 cells and a low toxicity against normal ARPE-19 cells (when two types of cells were co-cultured in the same culture media containing MMC-TG using a Transwell two-cell co-culture system). The nuclear targeting capabilities of both TG and MMC-TG were confirmed by CLSM and TEM measurements. TEM images further demonstrated the dynamic apoptosis of tumor cells induced by MMC-TG, which was observed to start from the lysis of cell nuclei. These results suggest that the as-synthesized MMC-TG is a good candidate as an antitumor drug carrier system, as it exhibited good tumorous nuclear targeting capability and a strong tumor suppression efficacy. Thus, this work suggests a new approach to the development of novel antitumor drugs.


N-isopropyl acrylamide) (LCHBPNIPAM) in aqueous solution. Here, we revealed a reversible self-assembly behavior of LCHBPNIPAM aqueous solution derived from temperature. By controlling the temperature of LCHBPNIPAM aqueous solution, we tune the morphology of the LCHBPNIPAM self-assemblies. When the solution temperature increased from the room temperature to the lower critical solution temperature of PNIPAM segments, LCHBPNIPAM self-assembled from multi-compartment vesicles into solid micelles. The morphology of LCHBPNIPAM self-assemblies changed from solid micelles to multi-compartment vesicles again when the temperature decreased back to the room temperature. The size presented, at first, an increase, and then a decrease, tendency in the heating-cooling process. The above thermally-triggered self-assembly behavior of LCHBPNIPAM aqueous solution was investigated by dynamic/static light scattering, transmission electron microscopy, atomic force microscopy, fluorescence spectroscopy, 1H nuclear magnetic resonance in D2O, and attenuated total reflectance Fourier transform infrared spectroscopy. These results indicated that LCHBPNIPAM aqueous solution presents a reversible self-assembly process. The controlled release behaviors of doxorubicin from the vesicles and micelles formed by LCHBPNIPAM further proved the feasibility of these self-assemblies as the stimulus-responsive drug delivery system.In this paper, we mainly described the reversible self-assembly of a backbone-thermoresponsive, long-chain, hyperbranched poly( Within the last decades, there has been increasing research interest in the various hyperbranched polymers because of their unique chemical and physical properties, such as highly-branched architectures with low viscosity and good solubility, as well as plenty of terminal groups for chemical modifications ,2,3,4. Pn (n ≥ 2) macromonomers amine , and NIPAM  were used as received. ca. 2 s. The frozen samples were subsequently freeze dried by slow warming to room temperature. The morphology was visualized using an AFM with a tapping mode and a Nanowizard II controller . Tip information: radius ≤ 33 mm, cantilever length 10 μm; width 100 μm; thickness 30 mm, resonant frequency 300 kHz, force constant 40 N/m. A Zetasizer Nano-ZS DLS  was used to determine the hydrodynamic diameter of self-assemblies. Each sample was kept at a predetermined temperature for 3 min before measurement without any filter. SLS analysis was performed on a DAWN HELEOS-II multi-angle light scattering detector  operated at 665 nm, using gallium-arsenic as the incident laser beam source. SLS data were collected at six different concentrations of the aggregates and 18 different angles for each concentration. The data were analyzed using the Zimm plot method on HELEOS-II Firmware 2.4.0.4 advanced software to determine gR. The emission spectra were recorded by FL  from 355 to 550 nm with an excitation wavelength at 335 nm. The existence of hydrogen bonding in the self-assembly process of the LCHBPs solutions were processed by FTIR-ATR spectra. A solution of the sample in either THF or water was placed in the liquid cell, and the spectra of the solutions were recorded.The size and morphology of the micelles with different polymer concentrations 0.02 mg/mL were revealed by TEM  at an acceleration voltage of 80 kV. The samples were prepared according to the literature ,40,41,42zD value of LCHBPNIAPM aqueous solutions with a constant polymer concentration of 0.2 mg/mL were recorded under different temperature conditions. Sample cells were thermostated with an internal constant temperature controller. The temperature ramp was set at 1 °C/min. The LCST values of the LCHBPNIPAM were defined as the suddenly increasing of zD value during the heating process.The LCST of LCHBPNIPAM was determined by Zetasizer Nano-ZS DLS . The The guest encapsulation of LCHBPNIPAM was measured by FL  using ANS (0.05 mM) as guest molecule in a buffer solution with ionic strength equal to 0.1 mol/L. Typically, the LCHBPNIPAM solution was diluted step-by-step to various desired concentrations (from 0.2 to 2.0 mg/mL) using different guest molecule solutions. All solutions were maintained for more than 12 h to ensure the binding equilibrium and then stirred prior to measurement.Mn 8000–14,000) against 1000 mL pure water at 20 °C with 300 r/min of stirring. Pure water was renewed for six times within 12 h (every 2 h). The final drug loading polymer aqueous had been lyophilized for use. For the release of DOX, the DOX-loaded polymers were dissolved in 5 mL of phosphate-buffered solutions (PBS) (1mg/mL) with different temperature (20 and 37 °C) and transferred into dialysis bags with a molecular weight cut off of 3500. Dialysis bags were then put into 30 mL PBS solutions for release. At given time intervals, 4 mL of aliquot was taken out to measure the DOX concentration in the dialysate with a UV–VIS spectrophotometer. Additionally, 4 mL of the corresponding fresh PBS solution was added after each sampling to ensure that the total volume of the buffer solution remained constant. The cumulative release was calculated by using Equation (2) as follows:W0 (mg) is weight of drug in the polymer; nC (mg/mL) is the concentration of DOX in buffer solution, which was withdrawn for n times, n−C1 (mg/mL) is the concentration of DOX in buffer solution, which was withdrawn for n − 1 times.The encapsulation and controlled release experiments of DOX were shown as follows. LCHBPNIPAM (20.0 mg) was dissolved in DMF (2.0 mL) and stirred for 2 h. Then DOX·HCl (6.0 mg) was dissolved in mixture solutions. Then 15 μL triethylamine was added dropwise to the solution and the mixture was stirred 12 h to reach equilibration. The unloaded free drug and the salt produced by neutralization reaction were removed by dialysis using a dialysis tube  was obtained by the click chemistry of the AB2 macromonomer  according to our previously work [zD) values at 35 °C (LCST point) (1H NMR (in D2O) measurements were conducted to obtain deeper insight into the self-assembly morphology and size.The resulting polymer LCHBPNIPAM  B was easZD) ranging from 264 to 637 nm were formed as the temperature was raised from 20 to 65 °C, revealing the occurrence of thermally-induced self-assembly [ZD value came back to 258 nm as the solution cooling to 20 °C. On the other hand, the change of zeta-potential of LCHBPNIPAM self-assemblies also reflected the reversibility of the self-assembly behaviors (ca. 35 °C) corresponding to the size transition. The absolute values of Zeta-potential showed an drastic decreasing from −0.64 to −23.2 mV, and then reducing to −3.58 mV, corresponding to the temperature increasing from 20 to 65 °C and decreasing to 20 °C, respectively. The vs. temperature is that the ionization of secondary amine group of PNIPAM chains is an endothermic process, so increasing the solution temperature may enhance the ionization behavior, further leading to the change of zeta potential as temperature. Additionally, increasing the temperature can promote the coalescence of self-assemblies, and further increase the surface negative charge density of self-assemblies. To further confirm the reversibility of the self-assembly process, we utilized a combination of SLS and DLS techniques to discriminate the inner structure of LCHBPNIPAM self-assemblies at initial and final states, respectively. It is known that the gR/hR value can predict the particle morphology [gR/hR value of 0.774, while a thin-layer hollow sphere of 1.00. The ratio gR/hR is useful for analyzing the structure of a nano-sized particle, since ratios close to 1.0 indicate that a hollow particle has been formed [gR/hR values of LCHBPNIPAM self-assemblies were 0.96 and 1.07 at initial and final states, respectively. Therefore, the gR/hR values 0.96 and 1.07 indicated a vesicular structure with a hollow cavity. The gR/hR ratio for the micelles is equal to 1.0. This supports the TEM observation of hollow characteristics. Furthermore, the results of SLS confirmed the reversibility of self-assembly process and the formation of multi-compartment vesicles.DLS was first used to study the size of LCHBPNIPAM in aqueous solution as shown in assembly . Howeverehaviors . It was rphology . For exan formed ,53. As savD) of 194 nm at 20 °C  in the emission spectrum was very sensitive to the polarity of the medium surrounding pyrene molecules [I3/I1 curves, indicating the formation of self-assemblies. Thus, the I3/I1 value of the pyrene emission spectra versus the logarithm of the LCHBPNIPAM concentration was 0.00675 mg/mL, as shown in max = 435 nm from 35 to 55 °C indicated that the releasing of the 1,8-ANS molecules into aqueous surroundings accompanied with the thermal collapsing of PNIPAM segments [max value of ANS increased with increasing LCHBPNIPAM concentration. The above results indicated that ANS guests were encapsulated into LCHBPNIPAM self-assemblies according to our previous work [FS spectroscopy was employed to further confirm the formation, reversibility and encapsulation functionality of LCHBPNIPAM self-assemblies. The critical aggregation concentration (CAC), which acted as a key parameter to quantitatively confirmation whether the micelles had been formed , was estolecules . The CACsegments . The intous work ,59.These above-mentioned results suggested that the thermo-induced reversible self-assembly of LCHBPNIPAM did, indeed, occur. Herein, the possible self-assembly mechanism was proposed. The hydrophilic PNIPAM segments form multi-compartment vesicles, membrane, and coronas, respectively , while t2O under different temperatures were compared, one significant change could be seen in the absorbance peak of the C=O groups. The peak at about 1646 cm−1 in THF  and solid micelles (above LCST) could be utilized as a feasible medium for encapsulation and release of drug molecules . Thus, Dof 0.990 B. This rN-isopropyl acrylamide) segments onto long chain hyperbranched polymer (LCHBPNIPAM) backbone can induce a reversible self-assembly. During the heating-cooling process, the morphology of LCHBPNIPAM self-assemblies changed from multi-compartment vesicles to solid micelles and back to multi-compartment vesicles again, and the size underwent a first increase and then decrease. The intermolecular hydrogen bonding plays an important role in forming various self-assembly structures of LCHBPNIPAM at different temperatures. The release rate of Doxorubicin from different LCHBPNIPAM self-assemblies can be effectively controlled. Therefore, these research results may be helpful to extend the application of long-chain hyperbranched polymers in drug delivery system.In conclusion, the introduction of thermoresponsive poly(